In this proposal we focus on the use of dendritic cells (DCs) to induce anti-tumor immunity in patients with advanced melanoma. Because of the manner in which they process protein, express co-stimulatory molecules, and elaborate cytokines that support the development of catalytic lymphocyte (CTL) responses, DCs are uniquely capable of antigen presentation. They appear to be ideal vehicles for delivery of antigen in tumor vaccination strategies. We have performed studies on the production of DCs in vitro from peripheral blood precursors, and have demonstrated our ability to reliably produce DCs in high fields by culturing PBMCs obtained via leukapheresis. Phenotypic analysis have shown that these cells express a profile of cell-surface markers characteristic of DCs, and functional studies demonstrate that they can be employed in vitro to routinely generate CTLs against antigens of choice. Concurrently we have utilized DCs to study antigen presentation of melanoma-specific antigens in vitro. Gp100 is a melanocyte and melanoma-specific marker that is immunogenic in melanoma patients, and preliminary studies suggest this antigen may have therapeutic value as a vaccine. We hypothesize that we can manipulate the molecular events controlling presentation of the gp100 tumor antigen by DCs. We verified that specific substitutions of the gp100 280 epitope result in peptides that bind to HLA-A2 with higher affinity than the native epitope. We chose the 280-9V epitope for further study, and showed that DCs pulsed with the modified epitope can stimulate CTLs specific for native 280 both in vitro and in vivo in a model system using an HLA-A2 transgenic mouse. Based on these studies, we propose to utilize our methods of DC manufacture to deliver the 280-9V transgenic mouse. Based on these studies, we propose to utilize our methods of DC manufacture to deliver the 280-9V modified gp100 epitope to patients with metastatic melanoma. Rigorous immunologic monitoring will be carried out. We will use CTL assays and Elispot assays to quantitatively determine levels of peptide- specific CD8+T cells. We also propose to isolate tumor infiltrating lymphocytes from patients following therapy, and to closely examine post-treatment tumor biopsy specimens with histopathologic and immunohistochemical methods for evidence of immune evasion. Concomitant with the execution of the trial of peptide-pulsed DC we will carry out a trial of DC therapy with adenovirus-modified DCs. Our final objective is the comparison of immunologic data obtained from the two trials.